Semester

Fall

Date of Graduation

2020

Document Type

Dissertation

Degree Type

PhD

College

Davis College of Agriculture, Natural Resources and Design

Department

Animal and Nutritional Sciences

Committee Chair

Scott Bowdridge

Committee Member

Mariette Barbier

Committee Member

Kimberly Barnes

Committee Member

Jennifer Franko

Committee Member

Jianbo Yao

Abstract

Globally, the small ruminant industry loses hundreds of millions of dollars due to parasitism by the gastrointestinal nematode Haemonchus contortus. This parasite feeds on blood in the host’s abomasum leading to disease including anemia, hypoproteinemia, lethargy, and death. There have been exhaustive efforts to manage this parasite including different management applications, anthelmintic treatment, and now integration of resistance genetics into a flock. Like antibiotics, overuse of anthelmintics has led to resistance of H. contortus to these drugs. Therefore, other methods of control are the main focuses of many research initiatives. St. Croix are a resistant breed of sheep that mount a Th2 immune response to third stage larvae (L3), preventing establishment of the parasite. Suffolk are susceptible to infection. In these studies, antibody from St. Croix and Suffolk were used to screen for H. contortus protein-sheep antibody binding that may contribute to the St. Croix immune response. This was carried out using immunoblotting technique and identified a 35 kDa H. contortus L3 protein bound by St. Croix serum, but not by Suffolk serum. This protein was identified via Mass Spec as tropomyosin domain containing protein. Tropomyosin has been used in several parasite vaccine trials and is also the main allergen of several arthropods including shrimp and cockroaches. These studies led to the continued investigation of tropomyosin as a putative vaccine candidate in sheep. Peptides were selected and synthesized based on University of Nebraska epitope prediction software and from a study that used tropomyosin peptides in proliferation assays of CD4+ cells isolated from patients allergic to shrimp. After receiving these peptides, enzyme linked immunosorbent assays (ELISA) were performed to determine if there was a breed or infection effect on antibody specificity to these peptides. There were no differences in antibody binding between naïve or primed (previously infected), St. Croix or Suffolk (Nebraska: P= 0.3889, 54: P=0.1674, 66: P= 0.4110, 80: P= 0.0527, 81: P= 0.3878). The second set of peptides was selected from the results of an epitope mapping experiment using overlapping Hc tropomyosin peptides screened with primed St. Croix antibody. Four peptides were selected from this experiment and were synthesized, a set with and without conjugation to keyhole limpet hemocyanin (KLH) for a vaccine trial. ELISA were performed with these peptides and showed no difference between groups (Peptide 1: P= 0.2501, Peptide 2: P= 0.0732, Peptide 3: P= 0.0900, Peptide 4: P= 0.3250). However, results based on the epitope mapping experiment provided better expectations for these peptides in a vaccine trial. The vaccine trial 25 weeks and included 4 groups- 1) vaccinated (peptide cocktail of 62.5 µg of each peptide + Montanide ISA 61 VG adjuvant), 2) adjuvant only, 3) infection only, and 4) naïve. Vaccinated and adjuvant only received an initial vaccination and a booster 4 weeks later. Six weeks later, animals in groups 1, 2, and 3 were challenged with 10,000 L3 H. contortus. The animals were dewormed 7 weeks later and rested for two weeks followed by a second challenge of 10,000 L3. Five weeks later, all animals were sacrificed. There were no differences in weekly or final infection parameters (fecal egg count: P= 0.8744, packed cell volume: P= 0.7532, adult worm burden: P=0.8189). However, the vaccinated group produced specific antibody to the peptide cocktail based on ELISA results. Gene expression revealed an adjuvant effect – increasing il17a expression. Although the vaccine was not protective, this experiment showed that Suffolk are able to respond to a vaccine and produce specific antibody to specific peptides. These experiments also provide a roadmap for future H. contortus vaccine development experiments.

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